Aryltetralin Lignan Glycosides From the Leaves of Viburnum cylindricum and Their Enzyme Inhibitory Activity

ABSTRACT

Viburnum cylindricum Buch. ‐Ham. ex D. Don is a commonly used medicinal Viburnum species containing abundant bioactive secondary metabolites. In this study, 14 aryltetralin lignan glycosides, including three previously undescribed ones, viburindrosides A–C (1–3), along with two rare variable 9‐O‐2′‐aryltetralin lignan glycosides (13 and 14), were isolated from V. cylindricum leaves by utilizing a liquid chromatography‐tandem mass spectrometry‐guided strategy. The structures of these compounds were elucidated by spectroscopic data, experimental and calculated electronic circular dichroism spectra, and chemical methods. In addition, the activity assessment results indicated that among all isolates, compounds 13 and 14 exhibited the strongest α‐glucosidase inhibitory activity with the half‐maximal inhibitory concentration (IC₅₀) values of 7.69 and 7.25 µM, respectively, while compounds 5 and 7 showed the potent urease inhibitory activity with the IC₅₀ values of 16.01 and 15.23 µM, respectively. Furthermore, molecular docking studies provided insights into the binding interactions of the four active compounds with α‐glucosidase and urease, respectively.

1. Introduction

Lignans constitute a substantial class of naturally occurring phenolic compounds, derived from the oxidative coupling of two phenylpropanoid (C6‐C3) units. In nature, lignans primarily exist as aglycones, with a minor proportion linked to sugars to form glycosides [1]. Aryltetralin lignans are a prominent lignan type characterized by the tricyclic framework (C₈‐C_8’ and C₂‐C_7’ connected) with two aromatic rings and one non‐aromatic ring [2]. This leads to a variety of possible configurations in aryltetralin lignans. Many lignans in this class showed significant anticancer, antiviral, antimicrobial, and antioxidant functions [3, 4]. In our preliminary research, some lignan constituents exhibiting significant enzyme inhibitory activities were discovered from the Viburnum plants (Adoxaceae), such as V. macrocephalum f. keteleeri, and V. urceolatum Siebold et Zucc [5, 6].

V. cylindricum Buch. ‐Ham. ex D. Don is an evergreen shrub or small tree commonly found in the provinces of central to southwest China. The whole plant of V. cylindricum, including the roots, stems, leaves, and fruits, can be used for medicinal purposes to treat dysentery, gastroenteritis, coughs, stomatitis, urinary tract infections, and rheumatic pain [7]. Previous phytochemical studies on V. cylindricum have identified the presence of bioactive constituents, including lignan glycosides, triterpenoids, diterpenoids, iridoids, and phenolic acids [8, 9, 10, 11, 12]. These compounds collectively contribute to its broad pharmacological potential, exhibiting hypoglycemic, anti‐inflammatory, and antioxidative activities [8, 13, 14]. As part of our ongoing research on lignan constituents with enzyme inhibitory activity from Viburnum plants, we discovered that the extract of V. cylindricum leaves exhibits potential α‐glucosidase and urease inhibitory effects. However, its active constituents remain uncharacterized. The liquid chromatography‐tandem mass spectrometry (LC‐MS/MS)‐guided fractionation on V. cylindricum leaves led to the isolation of 3 new aryltetralin lignan glycosides, viburindrosides A–C (1–3), together with 11 known analogues (4−14) (Figure 1). In this study, the isolation, structural elucidation, and enzyme (α‐glucosidase and urease) inhibitory activity of these aryltetralin lignan glycosides were described.

FIGURE 1.

The structures of compounds 1–14.

2. Results and Discussion

The treated extract (Fr.3) of V. cylindricum leaves was analyzed by LC‐MS/MS. As shown in Figure S28, three new aryltetralin lignan glycosides (1–3) and eleven known analogs (4–14) were detected and isolated from Fr.3.

Viburindroside A (1) was isolated as a white amorphous powder. Its molecular formula was determined to be C₂₇H₃₆O₁₂ based on high‐resolution electrospray ionization MS (HRESIMS) data at m/z 575.2094 [M+Na]⁺ (calcd. for C₂₇H₃₆O₁₂Na⁺, 575.2104), indicating 10 degrees of unsaturation. The comprehensive analysis of the ¹H and ¹³C nuclear magnetic resonance (NMR) data (Table 1) indicated that compound 1 was an aryltetralin lignan glycoside. The ¹H NMR spectrum displayed the presence of a 1,3,4,6‐tetrasubstituted phenyl [δ _H 6.66 (1H, s, H‐2) and 6.30 (1H, s, H‐5)], a 1,3,4,5‐tetrasubstituted phenyl [δ _H 6.33 (2H, s, H‐2′,6′)], three methines [δ _H 4.29 (1H, d, J = 4.5 Hz, H‐7′), 1.99 (1H, m, H‐8′), and 1.86 (1H, m, H‐8)], three methylenes [δ _H 3.97 (1H, dd, J = 9.4, 4.7 Hz, H‐9′a), 3.40 (1H, m, H‐9a), 3.31 (1H, m, H‐9b), 2.87 (1H, m, H‐9′b), 2.86 (1H, dd, J = 17.1, 5.6 Hz, H‐7b), and 2.59 (1H, dd, J = 17.1, 10.6 Hz, H‐7a)], and three methoxyl groups [δ _H 3.73 (3H, s, 3‐OCH₃) and 3.65 (6H, s, 3′,5′‐OCH₃)]. The ¹³C NMR spectrum showed 27 carbon resonances, including two C6–C3 units (δ _C 126.4, 111.7,146.2, 144.2, 116.1, 131.6, 31.9, 32.8, and 63.0; 133.0, 108.4 × 2, 147.2 × 2, 133.9, 44.0, 41.0, and 67.8), three methoxyl groups (δ _C 56.3×2 and 55.4), and a hexose moiety (δ _C 103.0, 76.8, 76.7, 73.7, 70.0, and 60.9). The NMR data presented above closely resemble those of compound 9, except for a distinct difference in chemical shifts of C‐8 (△δ = 6.1 ppm), indicating that compound 1 is a diastereoisomer of 9. Acid hydrolysis of 1 yielded the d‐glucose, which was identified by high‐performance LC (HPLC) analysis of its chiral derivatives [5]. The large J value (7.7 Hz) of the anomeric proton at δ _H 4.13 suggested it to be β‐configuration. The ¹H‐¹H correlated spectroscopy spectrum (Figure 2) revealed the presence of a linkage as ⁷CH₂‐⁸CH(⁹CH₂)‐^8′CH(^9′CH₂)‐^7′CH. The heteronuclear multiple bond coherence (HMBC) correlations from H‐1′′ to C‐9′ (δ _C 67.8) indicated that the d‐glucose group was located at the C‐9′ position. The three methoxy groups were linked to C‐3, C‐3′, and C‐5′ positions, respectively, determined by the corresponding nuclear Overhauser effect spectroscopy (NOESY) cross‐peaks from 3‐OCH₃ to H‐2, from 3′‐OCH₃ to H‐2′, and from 5′‐OCH₃ to H‐6′. The correlations from H‐8′/8 to H‐2′/6′, and from H‐7′ to H₂‐9′ were observed in the NOESY spectrum (Figure 3). Thus, H‐8, H‐8′, and H‐7′ were determined to be β‐oriented, β‐oriented, and α‐oriented, respectively. The absolute configuration of 1 was established as 8S,7′S,8′R by a comparison between its experimental and calculated electronic circular dichroism (ECD) data (Figure 4). Finally, the structure of 1 was elucidated and named as viburindroside A.

TABLE 1.

¹H (600 MHz) and ¹³C (150 MHz) nuclear magnetic resonance (NMR) data of compounds 1–3 in DMSO‐d ₆ (δ in ppm, J in Hz).

No.	1		2		3
	δ H	δ C	δ H	δ C	δ H	δ C
1		126.4		126.2		128.4
2	6.66 (s)	111.7	6.66 (s)	111.6	6.53 (s)	106.7
3		146.2		146.2		146.9
4		144.2		144.3		137.3
5	6.30 (s)	116.1	6.26 (s)	116.1		146.4
6		131.6		131.2		125.1
7	2.86 (dd, 17.1, 5.6) 2.59 (dd, 17.1, 10.6)	31.9	2.83 (dd, 17.0, 5.8) 2.57 (dd, 17.0, 10.4)	31.7	2.62 (dd, 15.1, 4.4) 2.44 (dd, 15.1, 11.6)	32.4
8	1.86 (m)	32.8	1.85 (m)	33.4	1.49 (m)	39.1
9	3.40 (m) 3.31 (m)	63.0	3.42 (m) 3.31 (m)	62.9	3.47 (m) 3.31 (m)	64.0
1′		133.0		132.8		138.3
2′	6.33 (s)	108.4	6.27 (s)	108.4	6.75 (d, 2.0)	113.1
3′		147.2		147.2		147.0
4′		133.9		133.9		144.0
5′		147.2		147.2	6.57 (d, 8.1)	114.9
6′	6.33 (s)	108.4	6.27 (s)	108.4	6.31 (dd, 8.1, 2.0)	119.7
7′	4.29 (d, 4.5)	44.0	4.18 (d, 4.8)	44.3	4.27 (d, 5.6)	40.2
8′	1.99 (m)	41.0	2.08 (m)	40.4	1.94 (m)	44.5
9′	3.97 (dd, 9.4, 4.7) 2.87 (m)	67.8	3.51 (m) 3.41 (m)	68.7	3.67 (m) 3.25 (m)	69.7
1′′	4.13 (d, 7.7)	103.0	4.14 (d, 7.8)	103.0	4.16 (d, 7.8)	103.3
2′′	3.06 (m)	73.7	3.02 (m)	74.0	3.05 (m)	73.5
3′′	3.07 (m)	76.8	3.05 (m)	77.0	3.06 (m)	76.9
4′′	3.10 (m)	70.0	3.07 (m)	70.3	2.99 (m)	70.1
5′′	3.15 (m)	76.7	3.15 (m)	76.7	3.16 (m)	76.9
6′′	3.63 (m) 3.45 (m)	60.9	3.65 (m) 3.40 (m)	61.2	3.64 (dd, 11.5, 5.9) 3.42 (m)	61.1
3‐OCH3	3.73 (s)	55.4	3.73 (s)	55.4	3.76 (s)	55.7
5‐OCH3					3.26 (s)	58.8
3′‐OCH3	3.65 (s)	56.3	3.63 (s)	56.1	3.69 (s)	55.7
5′‐OCH3	3.65 (s)	56.3	3.63 (s)	56.1

FIGURE 2.

Key ¹H‐¹H correlated spectroscopy (COSY) and heteronuclear multiple bond coherence (HMBC) correlations of compounds 1–3.

FIGURE 3.

Key nuclear Overhauser effect spectroscopy (NOESY) correlations of compounds 1–3.

FIGURE 4.

Experimental and calculated electronic circular dichroism (ECD) spectra of 1–3.

Viburindroside B (2), obtained as an amorphous powder, was determined to have the molecular formula as C₂₇H₃₆O₁₂ by the sodium adduct ion at m/z 575.2091 [M+Na]⁺ (calcd. for C₂₇H₃₆O₁₂Na⁺, 575.2104) in the HRESIMS spectrum, corresponding to 10 indices of hydrogen deficiency. The ¹H NMR and ¹³C NMR data were almost identical with those of 1, indicating that 2 was a diastereoisomer of 1. The relative configurations of H‐8, H‐8′, and H‐7′ in 2 were assigned as α‐orientation, α‐orientation, and β‐orientation, respectively, based on the key NOESY correlations from H₂‐9 to H₂‐9′, from H₂‐8′/8 to H‐2′/6′, and from H‐7′ to H₂‐9′ (Figure 3). The calculated ECD curve of 8R,7′R,8′S‐2 was consistent with the experimental spectrum (Figure 4). Consequently, the structure of 2 was identified and named as viburindroside B.

The molecular formula of viburindroside C (3) was identified as C₂₇H₃₆O₁₂ based on the HRESIMS peak at m/z 575.2089 [M+Na]⁺ (calcd. for C₂₇H₃₆O₁₂Na⁺, 575.2104), requiring 10 degrees of unsaturation. The ¹H NMR and ¹³C NMR data of 3 were similar to those of 4, with the primary difference being the absence of signals from one methoxy group at the C‐5 position in 3. The sugar group was identified as d‐glucose through HPLC analysis of its chiral derivative, compared to those of standard d‐ and l‐glucose [5]. It was linked to C‐9′ (δ _C 69.7) position, as demonstrated by the HMBC correlation from the anomeric proton at δ _H 4.16 to C‐9′ (Figure 2). The relative configuration of 3 was found to be identical to that of 11 by the NOESY correlations (Figure 3) from H₂‐9 to H₂‐8/8′, and from both H‐8′ and H₂‐9 to H‐2′/6′. The 8R,7′S,8′R‐configuration of 3 was clarified by comparing its experimental ECD data with the calculated ECD spectrum (Figure 4). Therefore, the structure of 3 was determined and named as viburindroside C.

Compounds 4–14, through comparisons of their experimental data with those reported in the literatures, were identified as (+)‐isolariciresinol 9‐O‐β‐d‐glucopyranoside (4) [15, 16], (+)‐isolariciresinol 4‐O‐β‐d‐glucopyranoside (5) [17], (‐)‐isolariciresinol 3α‐O‐β‐d‐glucopyranoside (6) [16], (‐)‐isolariciresinol 4‐O‐β‐d‐glucopyranoside (7) [18], (‐)‐5′‐methoxyisolariciresinol 3a‐O‐β‐d‐glucopyranoside (8) [16, 19], (+)‐5′‐methoxyisolariciresinol 3a‐O‐β‐d‐glucopyranoside (9) [16], (‐)‐lyoniresinol 9‐O‐β‐d‐glucopyranoside (10) [20], (+)‐lyoniresinol 4‐yl‐β‐d‐glucopyranoside (11) [21], (+)‐lyoniresinol 9‐O‐β‐d‐glucopyranoside (12) [20], (‐)‐ovafolinin B‐9′‐O‐β‐d‐glucopyranoside (13) [22], and (+)‐ovafolinin B‐9′‐O‐β‐d‐glucopyranoside (14) [22], respectively. The ¹³C NMR data for compounds 4–12 were provided in the Supporting Information.

All the isolated aryltetralin lignan glycosides (1–14) were assessed for their inhibitory activities against α‐glucosidase and urease (Table 2). It was found that compounds 1–14 showed potent inhibitory activity against α‐glucosidase with half‐maximal inhibitory concentration (IC₅₀) values of 7.25–25.98 µM, which were stronger than the positive control acarbose (IC₅₀ = 29.71 µM). Among them, compounds 13 and 14 showed the most potent inhibitory effect on α‐glucosidase (IC₅₀ = 7.69 and 7.25 µM, respectively). Meanwhile, the urease inhibitory activities of compounds 1–14 were better than that of thiourea (IC₅₀ = 28.57 µM), with IC₅₀ values ranging from 15.23 to 27.34 µM. Notably, compounds 5 and 7 exhibited the most remarkable urease inhibitory effect with the IC₅₀ values of 16.01 and 15.23 µM, respectively. Preliminary structure‐activity relationship studies indicated that among the tested compounds 1–12, the configurations at C‐7′, C‐8, and C‐8′ positions exhibited negligible influence on the inhibitory activities of both enzymes, while the two aromatic ring units may be the crucial active moiety. For example, compounds 1 and 2 exhibited comparable α‐glucosidase and urease inhibition, as did compounds 4 and 6. In the α‐glucosidase inhibition assay, compounds 13 and 14 displayed the strongest inhibitory activity, likely attributable to their epoxy‐bridged structural fragments. Regarding urease inhibition, compounds 5 and 7 exhibited the most significant effects, presumably due to the glucose residue linked to the hydroxyl group on the phenyl ring. Notably, the introduction of a methoxy group at C‐5 substantially decreased urease inhibitory activity, as evidenced by compound 12. Molecular docking analysis of the most active compounds 13 and 14 with α‐glucosidase, as well as 5 and 7 with urease, is illustrated in Figure 5 and Table S4. Compound 13 could stably bind with α‐glucosidase (‐9.1 kcal/mol) by forming hydrogen bond interactions with residues Ser157, Pro312, Arg315, Asp352, and Gln353, as well as hydrophobic interactions with Tyr158, Asp242, His280, Ser311, Arg315, and Asn415 (Figure 5A). In addition, compound 14 built tighter bindings (‐10.7 kcal/mol) with the essential residues Glu277, Arg315, Asp242, and Arg442 in the active site of α‐glucosidase through hydrogen bond and alkyl/Pi‐alkyl interactions (Figure 5B). As seen in Figure 5C,D, the complexes urease‐5 (‐8.9 kcal/mol) and urease‐7 (‐8.8 kcal/mol) were stabilized mainly by hydrogen bond, Pi‐sulfur, alkyl/Pi‐alkyl, and other hydrophobic forces with residues Ala169, Cys321, Ala 365, and surrounding residues.

TABLE 2.

The in vitro α‐glucosidase and urease inhibitory activities of compounds 1–14 from V. cylindricum leaves.

Compound	α‐glucosidase IC50 (µM)	Urease IC50 (µM)	Compound	α‐glucosidase IC50 (µM)	Urease IC50 (µM)
1	19.72 ± 0.88e	25.87 ± 1.26b	9	24.76 ± 0.57bc	26.19 ± 0.73b
2	20.18 ± 0.54e	26.13 ± 0.92b	10	22.25 ± 0.42cd	26.78 ± 1.06b
3	21.03 ± 0.61d	26.90 ± 0.59b	11	25.98 ± 1.04b	25.81 ± 0.58b
4	25.80 ± 0.95b	26.85 ± 0.74b	12	24.59 ± 0.68bc	27.34 ± 0.84ab
5	23.08 ± 0.68c	16.01 ± 0.28c	13	7.69 ± 0.25f	24.95 ± 0.77b
6	24.14 ± 0.72bc	25.03 ± 0.45b	14	7.25 ± 0.17f	25.28 ± 0.65b
7	23.71 ± 0.49c	15.23 ± 0.37c	Acarbose	29.71 ± 1.35a
8	24.97 ± 0.76bc	25.73 ± 1.13b	Thiourea		28.57 ± 1.45a

IC₅₀ values were presented as means ± standard deviation (SD, n = 3). Different superscript letters for data in the same evaluation test indicated a significant difference (p < 0.05). Acarbose and thiourea were used as the positive control.

FIGURE 5.

Molecular docking simulations of compounds 13 and 14 with α‐glucosidase (A/B) as well as 5 and 7 with urease (C/D).

3. Conclusions

In summary, 14 aryltetralin lignan glycosides (1–14), including three new compounds, viburindrosides A‐C (1–3) and two known rare variants of 9‐O‐2′‐aryltetralin lignan glycosides (13 and 14), were isolated from V. cylindricum leaves guided by an LC‐MS/MS strategy. Their structures were elucidated by comprehensive spectroscopic data (NMR and HRESIMS) analysis. The absolute configurations of the previously undescribed aryltetralin lignan glycosides were confirmed by comparing the experimentally measured ECD with calculated curves. All the isolated compounds were evaluated for their α‐glucosidase and urease inhibitory activities. The results revealed that compounds 13 and 14 exhibited potent α‐glucosidase inhibitory activity, while compounds 5 and 7 displayed the strongest inhibitory activities against urease. The further molecular docking analysis demonstrated that compounds 13 and 14 formed strong bindings with the key residues in the active site of α‐glucosidase through hydrogen bond and hydrophobic interaction. Meanwhile, compounds 5 and 7 could stably interact with urease owing to the occurrence of hydrogen bonds, Pi‐sulfur, and other hydrophobic forces. This work provides a scientific foundation for the further development and utilization of V. cylindricum leaves with these active aryltetralin lignan glycosides.

4. Experimental

4.1. General Experimental Procedures

1D and 2D NMR spectra were performed on a Bruker AVANCE‐600 spectrometer (Bruker, Germany). Ultraviolet (UV) and infrared (IR) spectra were recorded using Varian Cary 5000 and 610/670 FTIR spectrometers (Varian, USA), respectively. LC‐MS/MS data were acquired by a Bruker MaXis high‐resolution time‐of‐flight instrument with a YMC‐Pack ODS‐AQ column (4.6 × 250 mm, 5 µm). ODS (50 µm, YMC, Japan), Sephadex LH‐20 (40–70 µm, GE Healthcare Biosciences AB, Sweden), and AB‐8 macroporous resin (Nankai Hecheng, China) were used for column chromatography (CC). Bioreagents were purchased from Sigma‐Aldrich (St. Louis, MO, USA).

4.2. Plant Material

The leaves of V. cylindricum were collected in September 2022 in Guiyang city, Guizhou province, China. The specimen (YZ‐202203) was identified by Associate Professor Judi Fan of Guizhou Medical University and deposited in the authors’ laboratory, Yangzhou University, China.

4.3. Extraction and Isolation

The dried V. cylindricum leaves (3.5 kg) were powdered and extracted with MeOH under reflux (24 L, 2 h × 3 times) to yield a MeOH extract (157 g). This extract was suspended in water and then loaded onto an AB‐8 macroporous adsorption resin column, eluting successively with H₂O and 80% EtOH. The treated 80% EtOH fraction (103 g) was loaded onto a silica gel column chromatography (CC) using CH₂Cl₂‒MeOH (50:1 to 1:1, v/v) to obtain six fractions (Fr.1‒Fr.6). After enzyme inhibitory activity testing (Table S2), one part of Fr.3 was used for LC‐MS/MS analysis (Figure S28) and another was subjected to an ODS column eluting with MeOH‐H₂O (5:95→50:50, v/v) to yield three fractions (Fr.3‐1−Fr.3‐3). Fr.3‐2 through Sephadex LH‐20 (MeOH) and HPLC led to the purification of compounds 1 (8 mg, t_R = 29.4 min, MeOH‐H₂O, 26:74, v/v), 2 (5 mg, t_R = 27.3 min, MeOH‐H₂O, 26:74, v/v), 3 (6 mg, t_R = 30.8 min, MeOH‐H₂O, 30:70, v/v), 4 (15 mg, t_R = 27.2 min, MeOH‐H₂O, 31:69, v/v), 5 (11 mg, t_R = 28.8 min, MeOH‐H₂O, 25:75, v/v), 6 (16 mg, t_R = 27.5 min, MeOH‐H₂O, 32:68, v/v), 7 (12 mg, t_R = 25.7 min, MeOH‐H₂O, 24:76, v/v), 8 (15 mg, t_R = 18.1 min, MeOH‐H₂O, 32:68, v/v), 9 (14 mg, t_R = 22.4 min, MeOH‐H₂O, 31:69, v/v), 10 (9 mg, t_R = 23.1 min, MeOH‐H₂O, 28:72, v/v), 11 (13 mg, t_R = 22.6 min, MeOH‐H₂O, 25:75, v/v), 12 (9 mg, t_R = 25.4 min, MeOH‐H₂O, 28:72, v/v), 13 (11 mg, t_R = 30.9 min, MeOH‐H₂O, 35:65, v/v), and 14 (8 mg, t_R = 32.0 min, MeOH‐H₂O, 35:65, v/v).

Viburindroside A (1): ${[\alpha ]}_D^{23}$ = −24.1 (c 0.15, MeOH); HRESIMS m/z 575.2094 [M+Na]⁺ (calcd. for C₂₇H₃₆O₁₂Na⁺, 575.2104); UV (MeOH) λ _max nm log (ε) 205 (4.13), 282 (3.14); IR (KBr) v _max 3398, 2934, 1615, 1460, 1217, 1115, 1021, 625 cm⁻¹; ¹H NMR (DMSO‐d ₆, 600 MHz) and ¹³C NMR (DMSO‐d ₆, 150 MHz), see Table 1.

Viburindroside B (2): ${[\alpha ]}_D^{23}$ = −18.7 (c 0.1, MeOH); HRESIMS m/z 575.2091 [M+Na]⁺ (calcd. for C₂₇H₃₆O₁₂Na⁺, 575.2104); UV (MeOH) λ _max nm log (ε) 210 (4.10), 283 (3.18); IR (KBr) v _max 3375, 2934, 1614, 1514, 1460,1243, 1113, 1035, 632 cm⁻¹; ¹H NMR (DMSO‐d ₆, 600 MHz) and ¹³C NMR (DMSO‐d ₆, 150 MHz), see Table 1.

Viburindroside C (3): ${[\alpha ]}_D^{23}$ = −28.3 (c 0.15, MeOH); HRESIMS m/z 575.2089 [M+Na]⁺ (calcd. for C₂₇H₃₆O₁₂Na⁺, 575.2104); UV (MeOH) λ _max nm log (ε) 205 (4.17), 283 (3.07); IR (KBr) v _max 3363, 2938, 1681, 1613, 1513, 1460, 1205, 1123, 1032, 633 cm⁻¹; ¹H NMR (DMSO‐d ₆, 600 MHz) and ¹³C NMR (DMSO‐d ₆, 150 MHz), see Table 1.

4.4. Acid Hydrolysis of 1–3

Compounds 1–3 were subjected to acid hydrolysis according to the previously reported procedures [5]. Specifically, compound 1 (2 mg) was hydrolyzed in 1.0 N HCl (3 mL) at 70°C for 2 h. The hydrolysate was partitioned with CHCl₃, and the aqueous layer was dried under N₂. The residue was derivatized with l‐cysteine methyl ester hydrochloride and isothiocyanate in pyridine to generate chiral thiazolidine derivatives. HPLC analysis (CH₃CN/H₂O/HCOOH 25:75:0.05, 1 mL/min) revealed d‐glucose in all compounds (t_R = 14.8 min), confirmed by comparison with l‐glucose standard (t_R = 13.8 min).

4.5. α‐Glucosidase Inhibitory Assay

The α‐glucosidase inhibitory tests were performed using a previously described method [23] with acarbose serving as the positive control. A total of 20 µL of α‐glucosidase solution, 90 µL of phosphate buffer solution (0.1 mol·L⁻¹, pH 6.8), and 30 µL of sample solutions at varying concentrations were added to a 96‐well plate. The resulting mixture was incubated at 37°C for 20 min. Subsequently, 40 µL of pNPG solution (5 mmol/L) was introduced, followed by an additional incubation at 37°C for 20 min. Finally, 70 µL of 1 mol/L sodium carbonate solution was added to terminate the reaction, and the absorbance of the sample was measured at 405 nm. Each compound was analyzed in triplicate to determine the IC₅₀ value.

4.6. Urease Inhibitory Assay

The urease inhibition activity screening was conducted using a previously described method with minor adjustments [23]. Briefly, 70 µL of urea, 15 µL of urease solution, 15 µL of HEPES buffer, and 15 µL of the test sample solution at varying concentrations were added. The mixture was thoroughly combined and incubated at 37°C for 30 min. Following this, 50 µL of phenol‐sodium nitroprusside solution and 50 µL of sodium hypochlorite solution were added in succession, with thorough mixing between each addition, and the incubation continued at 37°C for 30 min. Thiourea was included as a positive control. Absorbance was measured at a wavelength of 630 nm, with three replicates for each sample.

4.7. Molecular Docking Study

The structural formulae of tested compounds were drawn in Chem‐Draw Ultra 18.0, and their favorable 3D conformations were obtained in Chem3D 18.0. The protein database (https://www.rcsb.org/) was used to search for α‐glucosidase (PDB ID: 3A4A) and urease (PDB ID: 1E9Y). The interactions of the enzyme with the compound were simulated in Autodock Vina. The 2D and 3D visualizations of the complex with the lowest binding energies were generated and analyzed through PyMOL and Discovery Studio 2019 [4].

Author Contributions

Jia Chen: investigation, data analysis, and writing – original draft. Meng‐Ya Yang: investigation and validation. Hong‐Juan Zhou: software and visualization. Xue‐Jing Zhu: validation and software. Jian‐Hua Shao: funding acquisition, resources, and writing – review & editing. Chun‐Chao Zhao: conceptualization, project administration, funding acquisition, and writing‐original draft/review & editing.

Funding

This research was supported by the National Natural Science Foundation of China (31201563) and State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Education (Guangxi Normal University, CMEMR2023‐B06).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv70693‐sup‐0001‐SuppMat.pdf

Data Availability Statement

The data that support the findings of this study are available in the Supporting Information of this article.